Chem. Res. Toxicol. 1988, 1 , 25-27
25
Bleomycln Congeners Exhibiting Altered DNA Cleavage Specificity Sir: The bleomycins (BLM’s) are a family of structurally complex glycoprotein antibiotics possessing significant anticancer activity ( I ) . They have been demonstrated to effect DNA strand scission in a reaction that is both metal ion and oxygen dependent (2);this transformation is believed to form the basis for the therapeutic efficacy of the bleomycins. A number of studies have demonstrated that BLM-mediated DNA cleavage is sequence specific (3-5), but the basis for this phenomenon is not understood. Previous studies have demonstrated that for certain bleomycin congeners the sequence specificity of DNA strand scission can be altered by the choice of metal cofactor ( 6 4 ,and by methylation (9) or platination ( I O ) of the DNA substrate. By the use of a palindromic dodecanucleotide, it has been shown that the ratio of DNA modification at individual sites can vary from one BLM congener to another (11). However, with the exception of tallysomycin (5, 12, 13), which differs substantially in structure from the other bleomycin group antibiotics (14), there is no report of the alteration of sequence selectivity of DNA cleavage among BLM congeners. Reported herein is an analysis of DNA cleavage by seven BLM congeners that establishes the existence of significant differences in the patterns of DNA cleavage. This finding provides important insights concerning the structural factors that control sequence selectivity, further supports the representation of activated FeBLM as a high valent metal-oxo complex, and may bear relevance to the design of a BLM congener having an altered spectrum of antitumor activity. The sequence selectivity of DNA strand scission was studied using three DNA restriction fragments1 and seven Fe(II).BLM derivatives.2 The pattern of DNA strand scission observed3 is illustrated for Fe(II).BLM A2 and Fe(II).epi-BLM A2 in Figure 1 and analyzed for four of the congeners in Table I as a function of cleavage efficiency observed at each of the 16 possible constituent dinucleotides.* The overall pattern of DNA strand scission was the same as that reported previously (3-5) with the exception that 5‘-GA-3‘was also found to be a prominent cleavage site in this study. Consistent with earlier reports, the sequence selectivity of DNA cleavage mediated by BLM B2 (4,13)or deglyco-BLMA2 ( 1 7,19,20)were found to be essentially identical with that of BLM A> Also quite similar was the pattern of DNA cleavage found here for iso-BLM A2 (supplementary material, Table I). On the other hand, the remaining three congeners exTwo 5’-[32P]end labeled fragments were obtained from SV40 as described (8). A third fragment was obtained in analogous fashion from pBR322 by using Hind I11 and Eco RV. These were obtained a8 reported previously (8, 15-13. In a typical experiment, the 5’-end labeled DNA fragment (15 pM final DNA nucleotide concentration) was dissolved in 30 pL (total volume) of 20 mM sodium cacodylate, pH 7.4, containing 40 mM NaCl. Bleomycin and Fe(II)(NH,)2(S04)2(1.5 pM final concentrations) were added simultaneously from separate solutions to initiate the reactions, which were maintained at 25 OC for 2 min and then analyzed by polyacrylamide gel electrophoresis. Following densitometric analysis of the polyacrylamide gels, the results were normalized for total cleavage in each lane. The relative cleavage efficiency was the average amount of cleavage obtained at each of the 16 possible dinucleotides, expressed to the nearest 20%. The results were shown to vary no more than 10-15% in replicate experiments or over a wide range (5-150 pM) of DNA nucleotide concentration. The amount of each BLM congener employed was adjusted to provide bands over the same range of DNA fragment sizes in each case. Analysis was limited to those lanes that contained significant amounts of uncleaved DNA restriction fragment; direct analysis of the effect of BLM concentration on sequence specificity demonstrated that sequence selectivity was not a strong function of the extent of DNA cleavage (supplementary material, Figure 1). A total of 233 dinucleotide sites were analyzed. Each of the sites occurred 10-22 times, with the exception of 5’-CG-3‘,which, occurred only 7 times.
Bleomycin A2 :RI= H,R2= C O W z Decarbamoylbleomycin A, : R,, R2= H Isobleomycin
A2
R,= CONH2, R2= H
hibited significant differences in both site selectivity and cleavage efficiency at individual dinucleotide sites. For example, epibleomycin A2 cleaved DNA less efficiently than BLM A2 at a number of sequences including 5’-AA-3’, (Table I). Epi5’-AC-3’,5’-AT-3’,5’-TC-3’,and 5’-TT-3’ bleomycin A2 also cleaved some sequences with greater efficiency, notably 5’-TG-3’and 5’-CT-3’,the former of which was not cleaved by any of the other congeners studied. A number of significant differences are illustrated in Figure 1, which also underscores the large variability seen for 5‘-GG-3’sequences! Relative to BLM A2, decarbamoyl BLM A2 failed to mediate cleavage at 5‘-AA-3‘,5’-AC-3‘,or 5’-CT-3’sites, and exhibited diminished cleavage at most other dinucleotide cleavage sites. It was the only BLM congener that would not mediate cleavage at 5‘-CT-3’. The greatest differences observed involved bleomycinic acid. Total DNA cleavage by bleomycinic acid was only -35% of that mediated by BLM A2,6undoubtedly reflecting an overall decrease in DNA affinity by this congener.’ Fe(II).bleomycinic acid failed to cleave DNA at four sites utilized by Fe(II).BLM A2 and gave diminished cleavage at all the sites utilized in common. Dinucleotides starting with a pyrimidine were generally poor cleavage sites for bleomycinic acid (Table I). Interestingly, the strongest cleavage sites for bleomycinic acid were 5’-GT-3’ and 5’-GA-3’,rather than 5’-GT-3’and 5’-GC-3’. The data obtained here support a model of bleomycinDNA interaction in which the bithiazole and C-terminal substituent govern DNA affinity, while the structural domain containing the chelated metal ion cofactor determines the precise geometry and chemical reactivity of the metal binding domain of metallobleomycin. The present results demonstrate clearly that alteration of either structural domain of bleomycin can lead to altered DNA This figure illustrates the differences in cleavage patterns between Fe(II).BLM A, and Fe(II).epi-BLM A, over a subset of the restriction fragment that contained similar amounts of cleavage for the two congeners. While the extent of cleavage of the restriction fragments differed in this particular experiment, the cleavage patterns observed were shown to be consistent with those obtained by statistical analysis of many fragments4 and not to be a strong function of total DNA cleavage over the concentration ranges studied (supplementary material, Figure 1). Under conditions that utilized 5 pM FeBLM and 15 pM DNA nucleotide concentrations. The comparable values for Fe(II).epi-BLM A2 and Fe(II).iso-BLM A, were 80% and -50%, respectively, of those obtained with Fe(II).BLM A? Presumably, the absence of a positively charged C-substituent results in diminished DNA binding; the negatively charged carboxylate moiety at the C-terminus of bleomycinic acid might also interact unfavorably with DNA phosphodiesters.
0893-228~/88/2701-0025$01.50/0 0 1988 American Chemical Society
26 Chem. Res. Toxicol., Vol. 1, No. 1, 1988 1 2 3 4
3C G '\
c
5 6
Communications
lr
Acknowledgment. This work was supported by PHS Research Grants CA-27603 and CA-38544, awarded by the National Cancer Institute, DHHS. Supplementary Material Available: A table of relative DNA cleavage of Fe(I1)-bleomycin cogeners BLM B2, deglyco-BLM A2, and iso-BLM A, and a figure portraying the effect of bleomycin concentration on the sequence selectivity of DNA strand scission (3 pages). Ordering information is given on any current masthead page.
'
A A GT C
CCT
E/-
:: :--G
the action of a high valent metal-oxo complex than from diffusible oxygen radicals (2, 11).
7 8 91011
References
COG
H
1
T C
CCT +GA +GG
C-
Figure 1. Sequence selectivity of strand scission of a n SV40derived, [5'-32P]end labeled DNA fragment 127 base pairs in length:' lane 1,untreated DNA; lane 2, Maxam-Gilbert "G-lane" (18);lane 3 , 5 pM Fe(II); lane 4,5 pM Fe(I1) 1mM dithiothreitol (DTT); lane 5 , 5 pM Fe(II)*BLMA,; lane 6 , 5 pM Fe(II)*epi-BLM A,; lane 7, 10 p M Fe(I1)eepi-BLM A,; lane 8,5 p M Fe(II)*BLM A,; lane 9, 5 pM Fe(I1)-epi-BLM A, 1 m M DTT; lane 10, 10 p M Fe(I1)-epi-BLM A, + 1m M D?T; lane 11,5 pM Fe(I1)OBLM A, + 1 mM DTT.
+
+
Table I. Relative DNA Cleavage Efficiency of Fe(I1) Bleomycin Congenersa Fe(I1)ebleomycin derivative dinucleotide BLM epi-BLM decarbamoylbleomy(5'-XY-3') A2 BLM A2 cinic acid A2 M 20 0 0 0 AC 20 0 0 0 AG 0 0 0 0 AT 60 20 20 0 CA 0 0 0 0 cc 0 0 0 0 CG 0 0 0 0 CT 40 60 0 40 GA 80 60 60 60 GC 100 80 80 20 GG 60 80 40 20 GT 100 100 100 80 TA 40 20 20 20 TC 60 20 40 20 TG 0 60 0 0 TT 40 0 40 0 PUPU 40 35 25 20 PuPy 70 35 40 25 PyPu 25 35 5 5 40 20 15 5 PYPY ~
=See footnotes 3 and 4 for the methods employed for DNA cleavage and analysis of sequence selectivity.
cleavage specificity.8 Mechanistically, the observed changes in DNA cleavage selectivity as a function of alteration of BLM structure seem more likely to derive from Structurally,Fe(II)-bleomycinicacid differs from Fe(II).BLM A2 only at the C-terminus. Fe(II)*decarbamoylbleomycin A2,which lacks a putative metal ligand (21) present in Fe(I1)-BLMA2, is believed to have an altered coordination geometry as a result (11). Assuming that it has the same Fe(I1) ligands as BLM A2, the Fe(II).epi-BLM A2 complex should differ from that of Fe(II).BLM A2 only in the orientation of the propionamide moiety relative to the metal center.
(1) Sikic, B. I., Rozencweig, M., and Carter, S. K., Eds. (1985) Bleomycin Chemotherapy, Academic, Orlando. (2) Hecht, S. M. (1986) "DNA strand scission by activated bleomycin group antibiotics". Fed. PFOC. 45, 2784-2791. (3) DAndrea, A. D., and Haseltine, W. A. (1978) "Sequence specific cleavage of DNA by the antitumor antibiotics neocarzinostatin and bleomycin". PFOC. Natl. Acad. Sci. U.S.A. 75, 3608-3612. (4) Takeshita, M., Grollman, A. P., Ohtsubo, E., and Ohtsubo, H. (1978) "Interaction of bleomycin with DNA". PFOC. Natl. Acad. Sei. U.S.A. 75, 5983-5987. (5) Mirabelli, C. K., Huang, C.-H., and Crooke, S. T. (1983) "Role of deoxyribonucleic acid topology in altering the site/sequence specificity of cleavage of deoxyribonucleic acid by bleomycin and talisomycin". Biochemistry 22, 300-306. (6) Chang, L.-H., and Meares, C. F. (1984) "Cobalt-bleomycins and deoxyribonucleic acid: sequence-dependent interactions, action spectrum for nicking, and indifference to oxygen". Biochemistry 23,2268-2274. (7) Kuwahara, J., Suzuki, T., and Sugiura, Y. (1985) "Effective DNA cleavage by bleomycin-vanadium(1V) complex plus hydrogen peroxide". Biochem. Biophys. Res. Commun. 129, 368-374. (8) Ehrenfeld, G. M., Shipley, J. B., Heimbrook, D. C., Sugiyama, H., Long, E. C., van Boom, J. H., van der Marel, G. A., Oppenheimer, N. J., and Hecht, S. M. (1987) "Copper-dependent cleavage of DNA by Bleomycin". Biochemistry 26,931-942. (9) Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M. (1985) "DNA methylation diminishes bleomycin-mediated strand scission". Biochemistry 24, 5285-5289. (10) Mascharak, P. K., Sugiura, Y., Kuwahara, J., Suzuki, T., and Lippard, S. J. (1983) "Alteration and activation of sequence-specific cleavage of DNA by bleomycin in the presence of the antitumor drug cis-diamminedichloroplatinum(I1)". Proc. Natl. Acad. Sei. U.S.A. 80,6795-6798. (11) Sugiyama, H., Kilkuskie, R. E., Chang, L.-H., Ma, L.-T., Hecht, S. M., van der Marel, G. A,, and van Boom, J. H. (1986) "DNA strand scission by bleomycin: catalytic cleavage and strand selectivity". J. Am. Chem. SOC.108, 3852-3854. (12) Mirabelli, C., Mong, S., Huang, C.-H., and Crooke, S. T. (1982) "Comparison of bleomycin A, and talisomycin A specific fragmentation of linear duplex DNA". Biochem. Biophys. Res. Commun. 91,871-877. (13) Kross, J., Henner, W. D., Hecht, S. M., and Haseltine, W. A. (1982) "Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin, and tallysomycin". Biochemistry 21,4310-4318. (14) Konishi, M., Saito, K.-T., Numata, K.-T., Tsuno, T., Asama, K., Tsukiura, H., Naito, T., and Kawaguchi, H. (1977) "Tallysomycin, a new antitumor antibiotic complex related to bleomycin. 11. Structure determination of tallysomycins A and B". J. Antibiot. 30, 789-805. (15) Umezawa, H., Takahashi, Y .,Fujii, A., Saino, T., Shirai, T., and Takita, T. (1973) "Preparation of bleomycinic acid hydrolysis of bleomycin B2 by a Fusarium acylagmatine amidohydrolase". J . Antibiot. 26, 117-119. (16) Kunishima, M., Fujii, T., Nakayama, Y., Takita, T., and Umezawa, H. (1976) "Chemistry of bleomycin. XVI. Epibleomycin". J. Antibiot. 29, 853-856. (17) Sugiyama, H., Ehrenfeld, G. M., Shipley, J. B., Kilkuskie, R. E., Chang, L.-H., and Hecht, S. M. (1985) J. Nut. Prod. 48, 869-877. (18) Maxam, A. M., and Gilbert, W. (1980) "Sequencing end-labeled DNA with base-specific chemical cleavages". Methods Enzymol. 65,499-560. (19) Sugiura, Y., Suzuki, T., Otsuka, M., Kobayashi, S., Ohno, M., Takita, T., and Umezawa, H. (1983) "Synthetic analogs and
Communications biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism”. J. Biol. Chem. 258, 1328-1336. (20) Umezawa, H., Takita, T., Sugiura, Y., Otsuka, M., Kobayashi, S., and Ohno, M. (1984) ‘DNA-bleomycin interaction. Nucleotide sequence-specific binding and cleavage of DNA by bleomycin”. Tetrahedron 40, 501-509. (21) Oppenheimer, N. J., Rodriguez, L. O., and Hecht, S. M. (1979) ‘‘Structural studies of ‘active complex’ of bleomycin: assignment
Chem. Res. Toxicol., Vol. 1, No. 1, 1988 27 of ligands to the ferrous ion in a ferrous-bleomycin-carbon monoxide complex”. h o c . Natl. Acad. Sci. U.S.A. 76, 5616-5620.
Joshua B. Shipley, Sidney M. Hecht* Departments of Chemistry and Biology University of Virginia Charlottesville, Virginia 22901 Received September 19, 1987
